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Bioprinted Amniotic Stem Cells Aid Wound Repair



"Bioprinted Amniotic Fluid-Derived Stem Cells Accelerate Healing of Large Skin Wounds"

Current therapies for skin injuries such a serious burns rely on autologous skin grafts; a therapy which is limiting with regards to the number and size of donor sites, allografts; which suffer from the problem of immune rejection, the more recently developed non-cellular dermal substitutes; which are expensive and not cosmetically advantageous or lastly complex biological skin equivalents; which again suffers from pricing and immune rejection problems. However some recent developments show great promise, such as cell spraying and bioprinting technologies which allow for the deposition of cells and biomaterials into precise three-dimensional geometries in order to create anatomically correct structures. Mesenchymal stem cells (MSCs) are considered an attractive cell for use in such techniques due to their therapeutic potential for repair and regeneration of tissues damaged by injury or disease (Maxson et al. and Wang et al.), as are amniotic fluid-derived stem (AFS) cells due to their high proliferation capacity, multipotency, immunomodulatory activity, and lack of significant immunogenicity (De Coppi et al. and Moorefield et al.). In a recent study in Stem Cells Translational Medicine researchers from the laboratory of Shay Sokera at the Wake Forest Institute for Regenerative Medicine, Winston-Salem, North Carolina, USA have shown that while MSCs and AFS cells both function well in wound closure and re-epithelialisation, AFS cells function better partly through increased secretion of trophic factors (Skardala and Macka et al.).

MSCs and AFP cells were deposited through a bioprinting process; this entails the depositing of two layers of a fibrin-collagen gel containing cells in between layers of thrombin at wound sites. Analysis of wound healing in mice found that at 1 week, both bioprinted MSCs and AFS cells had a similar percentage of unclosed wounds (44% and 42% respectively) which was significantly higher than control mice treated with the gel used as the carrier for the cells only (77%). At two weeks MSC and AFS cell treated wounds again had a similar percentage of unclosed wounds (2% and 3% respectively) again significantly higher than control (13%). Wound contraction at one week was significantly higher for the MSCs and AFP cells (44% and 43% respectively), as compared to control (17%), but at two weeks only the MSC treated mice had significantly higher wound contraction (83%) than control (73%). However, re-epithelialisation was higher at week one (21% and 25%) and week two (89% and 84%) in MSCs and AFP cells as compared to control at week one and two (7% and 51% respectively).

Histological sections of skin samples taken at week two found well-defined, organized epidermal layers of the regenerated skin in the MSC and AFS cell treated mice, while the control animals exhibited poorly defined epidermal layering. Additionally, MSC and AFP cell treated wounds demonstrated noticeably thicker regenerating tissue with a greater number of blood vessels as compared to control at week one and two, although AFP cell treated wounds had a greater number of vessels which were also noticeably larger. Microvessel density (MVD) values were also higher in the MSC and AFS cells treated animals at week one although the average vessel diameter (AVD) was not as compared to control. However at week two, while MVD values were still significantly higher, the AVD value for AFS cell treated animals was significantly higher than control, suggesting stabilization and maturation of new vessels over time. Analysis of SMA staining, which highlights smooth muscle cells (SMCs), demonstrated that AFS cell treated wounds had high number of SMCs and complete blood vessel maturation, while MSCs showed high numbers of SMCs, but not complete blood vessel maturation and control mice had few SMA-stained SMCs. This was confirmed through the analysis of red blood cell (RBC) appearance in the regenerating tissue; AFS cell treated mice had few RBCs, while MSC treated and control mice had many RBCs in the regenerating area confirming the lack of a mature vascular system.

AFS cells and MSCs were tagged with GFP before use, allowing them to be tracked after bioprinting. Day 1 analysis found large numbers of GFP+ cells within the wound area, and this decreased to 50% at day 4 with few GFP+ cells observed at day 7, and none observed at day 14. Additionally, no migration of these cells into the underlying tissue was observed suggesting that the printed cells remained in the tissue but did not permanently integrate. This further suggests that the observed increases in wound closure and neovascularization in MSC and AFS cell treated wounds may be due to the secretion of trophic factors from these cells. This hypothesis was then tested through the study of growth factor secretory profiles of medium conditioned by MSCs and AFS cells. This found that AFS cell conditioned medium (CM) contained many growth factors (basic fibroblast growth factor (b-FGF), hepatocyte growth factor (HGF) and the insulin-like growth factor-binding protein superfamily) at a higher concentration than the MSC CM, although the MSC CM did express higher levels of vascular endothelial growth factor (VEGF). Finally, using the CM medium in migration assays to test for the activity of the angiogenic growth factors found that significantly higher numbers human umbilical vein endothelial cells (HUVECs) migrated in response to AFS cell CM when compared to non-conditioned medium.

These data point towards both MSCs and AFS cells as being optimal cell types for bioprinting towards a more effective treatment for burns and skin wounds. Both cell types led to quick closure of wounds while AFS cell use increased neovascularization and blood vessel maturation, likely due to the secretion of trophic factors, although there was no observed migration and integration of the GFP-tagged AFS cells and MSCs into the regenerated skin. The advantages using AFS cells are obvious; they have extended life in vitro (up to 250 passages)(De Coppi et al., Moorefield et al. and Delo et al.), are multipotential and may have increased differentiation and expansion potential compared with MSCs (Valli et al.), although this point may be mute if the primary role of ASC cells is the release of trophic factors, and show a degree of immunomodulatory activity (Moorefield et al.). With all this in mind, it is possible that bioprinter-mediated deposition of AFS cells could be highly important in a clinical setting with the potential for tissues and organs to be built from scratch.



De Coppi P et al.
Isolation of amniotic stem cell lines with potential for therapy.
Nat Biotechnol 2007;25:100–106.

Delo DM et al.
Amniotic fluid and placental stem cells.
Methods Enzymol 2006;419:426–438.

Kolambkar YM et al.
Chondrogenic differentiation of amniotic fluid derived stem cells.
J Mol Histol 2007;38:405– 413.

Maxson S et al.
Concise review: Role of mesenchymal stem cells in wound repair.
Stem Cells Translational Medicine 2012;1:142–149.

Moorefield EC al.
Cloned, CD117 selected human amniotic fluid stem cells are capable of modulating the immune response.
PLoS One 2011;6: e26535.

Valli A al.
Embryoid body formation of human amniotic fluid stem cells depends on mTOR.
Oncogene 2010;29: 966–977.

Wang X et al.
Bone marrow mesenchymal stem cells increase skin regeneration efficiency in skin and soft tissue expansion.
Expert Opin Biol Ther 2012;12:1129–1139.



Study originally appeared in Stem Cells Translational Medicine.

Correspondent Stuart P Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.